The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef has no known catalytic activity, and functions by interacting with multiple host cell signaling proteins, including the Src-family kinases Hck, Lyn, and c-Src. During the previous funding period, we established in vitro and cell-based screens to identify small molecules that target the Nef: Hck complex. These screens identified two classes of drug-like compounds with low/sub-micromolar activity against Nef-dependent HIV replication in vitro, providing the first demonstration that small molecules targeting an HIV accessory protein-host cell protein complex have antiretroviral activity. In this renewal application, we will pursue these compounds in terms of their activity against a broader range of HIV strains, including strains resistant to clinical anti- retroviral drugs, in primary human host cells for HIV. We will also explore synergy of these Nef-directed compounds with established anti-HIV therapeutics and investigate their molecular mechanisms of action. The second major goal of this project is to test the hypothesis that the Nef oligomerization interface is a rational target for Nef-directed anti-HIV therapy. Previous studies have established that Nef oligomerization contributes to host cell kinase activation, as well as viral and immune receptor downregulation. Our own recent work shows that Nef mutations that interfere with oligomerization also prevent Nef-mediated enhancement of HIV replication in cell culture. Small molecule inhibitors of Nef oligomerization may therefore block critical Nef functions in HIV- infected cells, thus providing new scaffolds for anti-HIV drug development. A novel fluorescence complementation assay developed during the previous funding period that reports Nef oligomerization in live cells will serve as the basis for development of a high-content, cell-based chemical library screen to identify such compounds.